Transcriptome and Proteome Exploration to Model Translation Efficiency and Protein Stability in Lactococcus lactis

This genome-scale study analysed the various parameters influencing protein levels in cells. To achieve this goal, the model bacterium Lactococcus lactis was grown at steady state in continuous cultures at different growth rates, and proteomic and transcriptomic data were thoroughly compared. Ratios of mRNA to protein were highly variable among proteins but also, for a given gene, between the different growth conditions. The modeling of cellular processes combined with a data fitting modeling approach allowed both translation efficiencies and degradation rates to be estimated for each protein in each growth condition. Estimated translational efficiencies and degradation rates strongly differed between proteins and were tested for their biological significance through statistical correlations with relevant parameters such as codon or amino acid bias. These efficiencies and degradation rates were not constant in all growth conditions and were inversely proportional to the growth rate, indicating a more efficient translation at low growth rate but an antagonistic higher rate of protein degradation. Estimated protein median half-lives ranged from 23 to 224 min, underlying the importance of protein degradation notably at low growth rates. The regulation of intracellular protein level was analysed through regulatory coefficient calculations, revealing a complex control depending on protein and growth conditions. The modeling approach enabled translational efficiencies and protein degradation rates to be estimated, two biological parameters extremely difficult to determine experimentally and generally lacking in bacteria. This method is generic and can now be extended to other environments and/or other micro-organisms

Multi-omics approach to study the growth efficiency and amino acid metabolism in Lactococcus lactis at various specific growth rates

<p>Abstract</p> <p>Background</p> <p><it>Lactococcus lactis </it>is recognised as a safe (GRAS) microorganism and has hence gained interest in numerous biotechnological approaches. As it is fastidious for several amino acids, optimization of processes which involve this organism requires a thorough understanding of its metabolic regulations during multisubstrate growth.</p> <p>Results</p> <p>Using glucose limited continuous cultivations, specific growth rate dependent metabolism of <it>L. lactis </it>including utilization of amino acids was studied based on extracellular metabolome, global transcriptome and proteome analysis. A new growth medium was designed with reduced amino acid concentrations to increase precision of measurements of consumption of amino acids. Consumption patterns were calculated for all 20 amino acids and measured carbon balance showed good fit of the data at all growth rates studied. It was observed that metabolism of <it>L. lactis </it>became more efficient with rising specific growth rate in the range 0.10 - 0.60 h^-1, indicated by 30% increase in biomass yield based on glucose consumption, 50% increase in efficiency of nitrogen use for biomass synthesis, and 40% reduction in energy spilling. The latter was realized by decrease in the overall product formation and higher efficiency of incorporation of amino acids into biomass. <it>L. lactis </it>global transcriptome and proteome profiles showed good correlation supporting the general idea of transcription level control of bacterial metabolism, but the data indicated that substrate transport systems together with lower part of glycolysis in <it>L. lactis </it>were presumably under allosteric control.</p> <p>Conclusions</p> <p>The current study demonstrates advantages of the usage of strictly controlled continuous cultivation methods combined with multi-omics approach for quantitative understanding of amino acid and energy metabolism of <it>L. lactis </it>which is a valuable new knowledge for development of balanced growth media, gene manipulations for desired product formation etc. Moreover, collected dataset is an excellent input for developing metabolic models.</p

Investigation of the adaptation of Lactococcus lactis to isoleucine starvation integrating dynamic transcriptome and proteome information

Background: Amino acid assimilation is crucial for bacteria and this is particularly true for Lactic Acid Bacteria (LAB) that are generally auxotroph for amino acids. The global response of the Lmodel Lactococcus lactis ssp. lactis was characterized during progressive isoleucine starvation in batch culture using a chemically defined medium in which isoleucine concentration was fixed so as to become the sole limiting nutriment. Dynamic analyses were performed using transcriptomic and proteomic approaches and the results were analysed conjointly with fermentation kinetic data. Results: The response was first deduced from transcriptomic analysis and corroborated by proteomic results. It occurred progressively and could be divided into three major mechanisms: (i) a global down-regulation of processes linked to bacterial growth and catabolism (transcription, translation, carbon metabolism and transport, pyrimidine and fatty acid metabolism), (ii) a specific positive response related to the limiting nutrient (activation of pathways of carbon or nitrogen metabolism and leading to isoleucine supply) and (iii) an unexpected oxidative stress response (positive regulation of aerobic metabolism, electron transport, thioredoxin metabolism and pyruvate dehydrogenase). The involvement of various regulatory mechanisms during this adaptation was analysed on the basis of transcriptomic data comparisons. The global regulator CodY seemed specifically dedicated to the regulation of isoleucine supply. Other regulations were massively related to growth rate and stringent response. Conclusion: This integrative biology approach provided an overview of the metabolic pathways involved during isoleucine starvation and their regulations. It has extended significantly the physiological understanding of the metabolism of L. lactis ssp. lactis. The approach can be generalised to other conditions and will contribute significantly to the identification of the biological processes involved in complex regulatory networks of micro-organisms

Regulering av energimetabolisme i enterococcus faecalis studert med transkriptom-, proteom- og metabolomanalyser

Lactic Acid Bacteria (LAB) are widely used as starter culture in food fermentation. Among LAB also pathogenic bacteria are found particular in enterococci and streptococci. Enterococcus faecalis is a gut commensal bacterium but certain isolates have been shown to be pathogenic while others are foodgrade bacteria in LAB fermented food commodities. E. faecalis ferments sugars through different pathways, resulting in homo- or mixed acid fermentation. In homolactic bacteria glucose is converted to lactate in an ATP producing reaction. In mixed acid fermentation, in addition to lactate production, glucose is also converted to acetate, acetoin, formate, ethanol and CO2. However, there is limited information regarding to regulation of the central energy metabolism of E. faecalis. The aim of this work was to extend our knowledge with respect to the central energy metabolism of E. faecalis by employing metabolite, transcriptome and proteome approaches. High performance liquid chromatography and gas chromatography were used for metabolite measurements. DNA microarray technology and two dimensional gel electrophoresis combined with mass spectrometry analysis were used in transcription and protein expression analysis, respectively. Combining these approaches has not been performed in metabolic analysis in E. faecalis and this should give an in-depth understanding about regulation of the central energy metabolism in E. faecalis. This work showed that in absence of ldh (lactate dehydrogenase) gene, E. faecalis metabolizes glucose to ethanol, formate and acetoin. The change from homolactic to mixed acid fermentation affected expression of several genes and proteins mostly involved in energy metabolism. These genes play an important role in the regulatory network controlling energy metabolism in E. faecalis including acetoin production, and NAD+/NADH ratio. Additional studies were carried out in order to investigate the mixed acid fermentation of wild-type E. faecalis in chemostat during steady state and glucose limiting growth. Growth at three different growth rates demonstrated that the bacterium responded differently depending on the growth rate. At the highest dilution rate (D=0.4 h-1) most of the glucose was converted to lactate while at the lowest dilution rate (D=0.05 h-1) it changed towards mixed acids fermentation. Interestingly, increased growth rate induced the transcription of the ldh gene while the amount of Ldh protein was more or less unaffected. The differences in glucose energy metabolism at different growth and pHs between E. faecalis and two other LAB (Streptococcus pyogenes and Lactococcus lactis) and their LDH negative mutants were also investigated. Of note, deletion of the ldh genes hardly affected the growth rate in chemically defined medium under microaerophilic conditions. Furthermore, deletion of ldh affected the ability for utilization of various substrates as a carbon source. The final study explored the effect of ascorbate on growth in the absence of glucose and showed that E. faecalis can grow on ascorbate. In summary, the work presented in this thesis gave new insights in regulation and strengthens our knowledge regarding the metabolic pathways of glucose fermentation through the metabolite analysis, regulation of transcription and protein expression.Melkesyrebakterier brukes som startkulturer i en rekke ulike gjæringsreaksjoner i forbindelse med produksjon av mat. Enkelte melkesyrebakterier har også evnen til å forårsake sykdom, og dette gjelder spesielt for enterokokker og streptokokker. Enterococcus faecalis er en kommensal tarmbakterie. Likevel finner man innenfor denne arten både patogene isolater såvel som stammer benyttet i fermentering av matvarer. E. faecalis bryter ned sukker gjennom flere ulike veier, med enten melkesyre (homolaktisk gjæring) eller en blanding av syrer (blandet syregjæring) som endeprodukt. Homolaktiske bakterier bryter ned glukose til melkesyre i en reaksjonskjede som produserer ATP. Ved blandet syregjæring av glukose produseres det i tillegg til melkesyre også eddiksyre, acetoin, maursyre, etanol og CO2. Det er imidlertid lite informasjon om reguleringen av energimetabolismen i E. faecalis tilgjengenlig. Målet med arbeidet bak denne avhandlingen har derfor vært å tilegne oss kunnskap om den sentrale energimetabolismen i E. faecalis ved hjelp av ulike metoder for å studere metabolitter, transkriptomet og proteomet. Væskekromatografi og gasskromatografi ble brukt til metabolittmålinger, mens DNA mikromatriseteknologi og to-dimensjonal gelelektroforese kombinert med massespektroskopi ble brukt til henholdsvis transkripsjon- og proteinanalyser. Kombinasjonen av disse metodene har ikke tidligere blitt brukt i metabolske studier av E. faecalis, og vil derfor forhåpentligvis gi en dypere forståelse av overgangen mellom homolaktisk- og blandet syregjæring. Våre studier viser at i fravær av ldh genet, som koder for laktatdehydrogenase, blir glukose brutt med til etanol, maursyre og acetoin. Denne overgangen fra homolaktisk til blandet syregjøring påvirker uttrykket av en rekke gener og proteiner involvert i energimetabolismen. Genene innehar viktige roller i det regulatoriske nettverket som kontrollerer energimetabolismen i E. faecalis, og inkluderer gener involvert i produksjon av acetoin og balansen mellom NAD+/NADH. Videre studier ble også gjort for å undersøke blandet syregjæring i villtype E. faecalis i kjemostat ved likevektstilstand og glukosebegrenset vekst. Vekst ved tre forskjellige veksthastigheter viste av bakterien responderer forskjellig avhengig av veksthastighet. Ved den høyeste fortynningshastigheten (D=0.4 h-1) ble det meste av glukosen omdannet til melkesyre, mens en endring i retning av blandet syrefermentering ble observert ved den laveste fortynningshastigheten (D=0.05 h-1). Interessant nok så førte økt veksthastighet til økt transkripsjon av ldh-genet, men mengden Ldh-protein var tilnærmet uendret. Forskjellene i nedbrytning av glukose ved forskjellige veksthastigheter og ved forskjellig pH mellom E. faecalis og to andre melkesyrebakterier (Streptococcus pyogenes and Lactococcus lactis) ble også undersøkt. Det er her verdt å merke seg at inaktivering av ldh genene hadde liten innvirkning på veksthastigheten til de ulike bakteriene i kjemisk definert medium under mikroaerofile vekstforhold. Inaktiveringen av ldh påvirket også bakterienes evne til å utnytte andre substrater enn glukose som karbonkilde. I det siste arbeidet i avhandlingen ble det vist at E. faecalis i fravær av glukose er istand til å vokse på askorbinsyre. Sett under ett har arbeidet som er presentert i denne avhandlingen, gjennom analyser av metabolitter, transkripsjonregulering og proteinuttrykk, gitt økt innsikt i reguleringen av og styrket vår kjennskap til veiene for nedbrytning av glukose.Norges Forskningrå

Comprendre l’adaptation de Lactococcus lactis par une\ud approche de biologie intégrative à l’échelle du génome

L’adaptation de Lactococcus lactis à différentes conditions de culture a été appréhendée grâce à une\ud démarche de biologie intégrative. Cette approche intègre les données issues de différents niveaux de\ud régulation et combine diverses techniques de mesure à l’échelle globale (transcriptome, protéome,\ud stabilité des ARN messagers) et locale (suivi des paramètres de culture). Plusieurs outils\ud mathématiques de modélisation (tels que la modélisation numérique et la modélisation statistique)\ud ont étés développés pour intégrer l’ensemble de ces données hétérogènes.\ud Une culture continue de L. lactis à différents taux de dilution a permis d’étudier l’influence du taux\ud de croissance sur la physiologie de la bactérie, un paramètre qui n’est jamais distingué de la réponse\ud au stress lors des études dynamiques de l’adaptation. La réponse à la variation du taux de croissance\ud implique majoritairement les fonctions associées à la biogenèse mais demeure extrêmement étendue\ud puisqu’elle affecte l’expression de 30 % des gènes de L. lactis. Cette réponse concerne les niveaux\ud d’ARN messagers et de protéines mais aussi les processus cellulaires majeurs que sont la\ud traduction, la dilution et la dégradation. Il a été montré, par une approche de modélisation, que les\ud efficacités de traduction et les vitesses de dégradation des protéines étaient en effet inversement\ud proportionnelles au taux de croissance. Au final, l’influence des différents processus cellulaires a pu\ud être quantifiée par des calculs de coefficients de contrôle.\ud L’imposition progressive d’une carence en isoleucine lors d’une culture discontinue en batch a\ud permis de caractériser la réponse, encore peu étudiée, de L. lactis à une carence en acide aminé.\ud L’adaptation à ce stress nutritionnel entraîne une vaste réorganisation de la physiologie cellulaire\ud qui se divise en trois types de réponses : une répression globale des principales fonctions\ud biologiques associées à la croissance, une réponse propre au stress imposé visant à lutter\ud spécifiquement contre la carence en isoleucine, ainsi qu’une activation inexpliquée de mécanismes\ud en lien avec le stress oxydatif. L’implication de différents mécanismes (réponse stringente,\ud mécanisme lié au taux de croissance, régulations par CodY, GlnR et CcpA) dans la régulation de\ud cette réponse a été évaluée par transcriptomique comparative.\ud Les déterminants majeurs des concentrations en protéines au sein de la cellule ont été recherchés\ud mathématiquement grâce à un algorithme de sélection de modèles de covariances. Le biais de\ud codons (CAI) s’est avéré être un paramètre majeur, plus important que les concentrations en ARN\ud messagers, suggérant l’existence d’un contrôle génétique prépondérant sur l’adaptation\ud transcriptionnelle. Enfin, il a pu être démontré que le degré d’implication des différents\ud déterminants varie en fonction du mode d’adaptation.\ud L’approche de biologie intégrative suivie au cours de cette thèse a permis une meilleure\ud compréhension des mécanismes d’adaptation de L. lactis et est aujourd’hui entièrement\ud généralisable à d’autres processus comme à d’autres microorganismes. ------------------------------------------------------------------------------------------------------------------------------------------------------------------------- A systems biology approach was implemented to study Lactococcus lactis adaptation to\ud various growing conditions. This method combines growth parameter monitoring and\ud genome-wide measurement technologies (transcriptome, proteome, messenger RNA\ud stability). Data from these diverse regulation levels were integrated thanks to mathematical\ud tools developed on purpose.\ud Growth rate influence on L. lactis physiology, which is never dissociated from stress\ud responses when studying dynamic adaptation processes, was analysed through continuous\ud culture at various growth rates. This widespread response mainly involves biogenesis-related\ud functions and affects the expression of 30 % of L. lactis genes. Both messenger RNA and\ud protein levels are modified but cellular processes such as translation, dilution and degradation\ud are also concerned. As a matter of fact, translation efficiency and protein degradation rates\ud were proved to be inversely proportional to growth rate by a modelling approach. Control\ud coefficient calculations enabled the quantification of cellular processes influences.\ud The dynamic response of L. lactis to isoleucine starvation was studied by the progressive\ud consumption of this amino-acid in a discontinuous batch fermentation. This poorly\ud characterized adaptation process triggers a wide reorganization of cellular physiology that\ud could be divided in three parts: a global repression of the main biological functions related to\ud growth, a response more specific to the encountered stress to struggle against isoleucine\ud starvation and an unexplained activation of oxidative stress-related cellular functions.\ud Comparative transcriptomics allowed the implication of various mechanisms to be quantified\ud in the regulation of this adaptation response (stringent response, growth rate adaptation\ud mechanism, CodY, GlnR and CcpA regulation).\ud The major biological determinants of protein intracellular concentration were mathematically\ud investigated thanks to a covariance model selection algorithm. Codons bias (CAI) was found\ud to be the most influent parameter, even more than mRNA concentrations, which suggests that\ud genetic control is stronger than transcriptional adaptation. The weight of the different\ud determinants was also found to depend on adaptation modes.\ud The systems biology approach applied in this work enabled a better understanding of L. lactis\ud adaptation mechanisms and will be entirely transposable to other cellular processes as well as\ud other microorganism

Systems biology of lactic acid bacteria: a critical review

Understanding the properties of a system as emerging from the interaction of well described parts is the most important goal of Systems Biology. Although in the practice of Lactic Acid Bacteria (LAB) physiology we most often think of the parts as the proteins and metabolites, a wider interpretation of what a part is can be useful. For example, different strains or species can be the parts of a community, or we could study only the chemical reactions as the parts of metabolism (and forgetting about the enzymes that catalyze them), as is done in flux balance analysis. As long as we have some understanding of the properties of these parts, we can investigate whether their interaction leads to novel or unanticipated behaviour of the system that they constitute

Metabolic shifts in microorganisms: the case of Lactococcus lactis

A commonly observed organismal response to changing growth rate is a metabolic shift from one mode of metabolism to another. This phenomenon is potentially interesting from a fundamental and industrial perspective because it can influence cellular choices and can limit the capacity of industrial microorganisms to channel nutrients to desired products. The mechanistic cause of the metabolic shift may vary between species, but the presence of such shifts from bacteria to man suggests functional relevance, which may be understood through an evolutionary perspective. One of the many existing hypotheses (reviewed in Chapter 2) states that protein investment costs affect the metabolic strategy employed, and that the implemented strategy is the result of a cost-benefit analysis. To test this experimentally, we performed a global multi-level analysis using the model lactic acid bacterium Lactococcus lactis subsp. cremoris MG1363, which shows a distinct, anaerobic version of the bacterial Crabtree/Warburg effect: at low growth rates it produces “mixed-acids” (acetate, formate and ethanol) and at high growth rates it produces predominantly lactate from glucose. We first standardized growth conditions and established an in vivo–like enzyme assay medium mimicking the intracellular environment for enzyme activity measurements of growing cells of L. lactis (Chapter 3). With standardized experimental procedures we characterized at multiple cellular levels, glucose-limited chemostat cultures of L. lactis at various growth rates. More than a threefold change in growth rate was accompanied by metabolic rerouting with, surprisingly, hardly any change in transcription, protein ratios, and enzyme activities (Chapter 4). Even ribosomal proteins, constituting a major investment of cellular machinery, scarcely changed. Thus, contrary to the original hypothesis, L. lactis displays a strategy where its central metabolism appears always prepared for high growth rate and it primarily employs the regulation of enzyme activity rather than alteration of gene expression. Only at the highest growth rate and during batch growth – conditions associated with glucose excess – we observed down-regulated stress protein levels and up-regulated glycolytic protein levels. We conclude from this that for glucose, transcription and protein expression largely follow a binary feast / famine logic in L. lactis. To delve deeper into the mechanism of regulation of the shift in L. lactis, we tested a mixed-acid fermentative lactose-utilizing L. lactis MG1363 derivative and showed that there is a strong positive correlation between glycolytic flux and the extent of homolactic fermentation: a correlation caused by metabolic regulation (Chapter 5). We subsequently provided new evidence for a causal relationship between the concentration of the glycolytic intermediate, fructose-1,6-bisphosphate (FBP) and the metabolic shift. We showed that 2,5-anhydromannitol, which converts to a non-metabolizable FBP analogue in vivo, almost doubles the flux towards lactate when taken up by the cells. In vitro the activating effect of the analogue on lactate dehydrogenase is similar to native FBP, whereas it had no effect on the enzyme phosphotransacetylase (part of the mixed-acid pathway). The activation concentration of the analogue, however, is much lower than normal intracellular FBP concentrations. This may imply that the activation of lactate dehydrogenase in vivo requires a much higher concentration of FBP, but this remains to be resolved. We subsequently put the regulatory relationships of glycolytic flux, FBP, the redox potential and allosteric effectors on enzymes of the glycolytic and downstream pathways together in a mathematical model to test and investigate whether these interactions can explain the metabolic shift (Chapter 6). Although the model was not able to consistently fit combined data from the chemostats at various dilution rates, and in vivo–NMR data of glucose pulsed non-growing cells, we found for the best fitted model that the parameters most influencing the metabolic shift were those involved in regulation by FBP and inorganic phosphate. In conclusion, L. lactis seems to be always prepared for high growth rate as it carries a high overcapacity of enzymes, a property retained even after evolving for 800 generations under constant environmental conditions. Moreover, its growth rate-related metabolic shift does not appear to be an outcome of growth-rate optimization with protein cost as a major driver. At the mechanistic level, the choice of the strategy is regulated via alterations in metabolite levels, with FBP (and probably phosphate) exerting a central role.</p